A considerable amount of work over the past two decades has established that ADH functions by recruiting proteinaceous water channels from cytoplasmic vesicles to the apical plasma membrane of collecting duct principal cells. Since the last Program Project renewal, several water channels (aquaporins), have been identified, and one of them, AQP2 is the long sought after vasopressin-sensitive water channel. This protein, together with other aquaporins that are present in diverse renal epithelial cell membranes, serves to regulate renal water reabsorption and to maintain body fluid homeostasis. The aquaporins represent an interesting family of functionally important proteins that are also of great cell biological interest because different family members have distinct intracellular trafficking and targeting characteristics. A major advance that will be extensively utilized in this renewal application is our successful reconstitution of the constitutive and regulated pathways of aquaporin 1 and aquaporin 2 membrane trafficking in transfected LLC-PK1 epithelial cells (referred to as LLC-AQP1 and LLC-AQP2 cells). The major objectives of this renewal are to exploit these novel in vitro systems to dissect mechanisms and pathways of regulated and constitutive aquaporin processing and trafficking, and to relate this information to the polarized delivery of aquaporins in vivo, as well as to the cellular biology of epithelial cell protein transport in general. Our Specific aims are: 1) To elucidate the pathways of intracellular processing, packaging and recycling of AQP2 using immunocytochemistry, membrane fractionation, pulse-chase radiolabeling and immunoprecipitation, cell-surface biotinylation and freeze-fracture electron microscopy in stably transfected cells. This pathway will be compared to the constitutive pathway followed by AQP1 in transfected cells: 2) To determine the targeting signals on aquaporins that are involved in constitutive versus regulated plasma membrane insertion of these proteins, using site-directed mutagenesis and chimeric proteins expressed in cultured cells: 3) To determine the interaction of AQP- containing vesicle with accessory proteins that are involved in regulated exo- and endocytosis, including GTP-ginding proteins, annexins, synaptic vesicle-like proteins, vesicle coat proteins and cytoskeletal- associated proteins.